PROJECT SUMMARY/ABSTRACT The periodontal complex, including cementum, periodontal ligament (PDL), and alveolar bone, is critical for tooth attachment and function. Periodontal diseases are among the most prevalent on earth, causing periodontal destruction and tooth loss, and affecting health and quality of life. Periodontal regeneration is possible, however, current therapies are unpredictable, few are truly regenerative, and many lack a biologic foundation. The path to regeneration remains unclear, in part, because origins and differentiation of cementoblasts (cells that produce cementum), and regulatory processes in cementogenesis, remain poorly understood. Bone sialoprotein (Ibsp gene; BSP protein) is a multifunctional extracellular matrix (ECM) protein associated with mineralized tissues, skeletal formation, and bone remodeling. Ibsp knockout (Ibsp-/-) mice feature absence of functional acellular cementum, PDL detachment, defective alveolar bone and cellular cementum mineralization, and alveolar bone resorption and tooth loss. The underlying mechanisms of BSP function remain unknown, although BSP harbors three functional domains, including a collagen-binding domain, polyglutamic acid (polyE) motifs that promote mineralization, and an arginine-glycine-aspartic acid (RGD) integrin-binding domain that initiates cell signaling. We propose that BSP is a unique candidate factor for studying cementum formation and alveolar bone healing because it is selectively expressed, essential for proper function, and operates by non-redundant mechanism(s) distinct from other growth factors. Based on our preliminary data, our hypotheses are that cementoblasts are BSP-expressing ectomesenchymal cells distinct from osteoblasts; BSP signals bone cells via the RGD domain in bone remodeling and directs mineral deposition onto collagen fibrils via the collagen-binding domain; and BSP promotes alveolar bone healing. These hypotheses will be tested by 3 specific aims: (1) To define the origin and transcriptome of cementoblasts using conditional ablation of Ibsp from ectomesenchymal vs. epithelial cell populations, and use endogenous yellow fluorescent protein expression in Ibsp-topaz mice to ex vivo purify cementoblasts by fluorescence-activated cell sorting (FACS) and perform transcriptomic analysis; (2) To analyze the mechanism by which BSP functions in bones and teeth by defining the binding site of BSP on collagen and analyzing inactivation of BSP RGD and collagen-binding domains in cementoblasts in vitro and genetically engineered mouse lines in vivo; and (3) To evaluate the role of BSP in alveolar bone repair using a molar socket healing model in mice and an osteotomy healing model in human subjects to map BSP expression, and determine healing outcomes in mice when BSP is ablated or specific functional domains have been inactivated.